The Ebola Virus Matrix Protein Deeply Penetrates the Plasma Membrane: An Important Step in Viral Egress

Abstract

Ebola virus, from the Filoviridae family has a high fatality rate in humans and nonhuman primates and to date, to the best of our knowledge, has no FDA approved vaccines or therapeutics. Viral protein 40 (VP40) is the major Ebola virus matrix protein that regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of VP40 membrane binding that are important for viral release remain to be elucidated. In this study, we used fluorescence quenching of a tryptophan on the membrane-binding interface with brominated lipids along with mutagenesis of VP40 to understand the depth of membrane penetration into lipid bilayers. Experimental results indicate that VP40 penetrates 8.1 Å into the hydrocarbon core of the plasma membrane bilayer. VP40 also induces substantial changes to membrane curvature as it tubulates liposomes and induces vesiculation into giant unilamellar vesicles, effects that are abrogated by hydrophobic mutations. This is a critical step in viral egress as cellular assays demonstrate that hydrophobic residues that penetrate deeply into the plasma membrane are essential for plasma membrane localization and virus-like particle formation and release from cells.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine

“…However, the majority of this previous work predated knowledge on purification conditions and mutations that may influence VP40 dimer, monomer, or octameric ring structure. In fact, previous work from the Stahelin lab using a GST-VP40 expression system with subsequent cleavage of the GST tag (13,20) demonstrated purification of monomeric VP40 identified by chemical crosslinking (E. Adu-Gyamfi and R.V. Stahelin, unpublished data).…”

“…A number of earlier studies have examined the role of PS in lipid vesicle binding and cellular assembly of VP40 demonstrating an important role for PS in membrane binding and membrane curvature changes (13,19,20,31,32) as well as a critical requirement of plasma membrane PS levels in VLP formation (27). However, the majority of this previous work predated knowledge on purification conditions and mutations that may influence VP40 dimer, monomer, or octameric ring structure.…”

“…The first X-ray structure of VP40 (17) demonstrated that VP40 harbored an Nterminal domain that has been shown to mediate VP40 oligomerization (1,7,18) and a C-terminal domain that can interact with anionic membranes (1,13,(19)(20)(21)(22). Additionally, VP40 harbors a ~43 amino acid long Nterminal region that has not been resolved in crystal structures.…”

“…While the molecular basis of VP40 association with the anionic inner leaflet of the PM is unknown, it is clear that both electrostatic (19) and hydrophobic interactions (10,13) play an important role. Hydrophobic residues in the Cterminal domain are critical to localization, membrane penetration, and budding (13) while interactions with phosphatidylserine (PS) have been shown to be important for association with lipid vesicles in vitro (19,20,22,27) and budding from the plasma membrane of mammalian cells (27).…”

The Ebola Virus Matrix Protein VP40 Interacts With Several Host Protein Networks to Facilitate Viral Replication

“…This suggested that perhaps an L-domain in one of the other viral proteins may be contributing or possibly VP40 may achieve scission without the aid of the host cell. To the credit of the latter hypothesis, VP40 alone has been shown to mediate budding and scission into synthetic vesicles recapitulating plasma membrane lipid composition [30,31]. However, it was revealed recently that VP40 possesses a third L-domain, a YPxL/I motif , which has been shown to mediate interactions with the early ESCRT protein Alix [12••].…”